The present invention relates generally to methods of multidimensional chemical analysis. More specifically, the system and method of this invention pertains to on-line coupling of liquid capillary separations with mass spectrometric detection.
Most complex biomedical and environmental samples require application of complimentary mulitdimensional analysis methods to compensate for sample and matrix interferences, and to obtain efficient analyte separation/purification in order to provide reliable qualitative (molecular weight and structural elucidation) and quantitative results. Mass spectrometers have been used extensively as detectors for various separation methods. For example, gas chromatography/mass spectrometry provided a breakthrough in hyphenated methods of chemical analysis.
Until recent years, most mass spectral methods produced strong molecular fragmentation and were not applicable for analysis of complex high molecular weight compounds. The recent introduction of electrospray ionization (ESI), atmospheric pressure ionization (API), atmospheric pressure chemical ionization, and matrix assisted laser desorption ionization (MALDI) changed the status of mass spectrometry (MS). These methods provide minimal fragmentation and high sensitivity for analysis of a wide variety of fragile and nonvolatile compounds. MALDI has been applied to the analysis of peptides, proteins, lipids, oligosaccharides, oligonucleotides, dyes, and synthetic polymers. Hillencamp, et al., Anal. Chem. 63: 1193A-1202A (1991). Glycoproteins with a large proportion of carbohydrate, normally refractory to mass spectral analysis, produce intense spectra when analyzed by MALDI-MS.
MALDI is often combined with time-of-flight (TOF) mass spectrometry, providing detection of molecular mass up to 106 Da and sample size in the atomole range.
Various off-line combinations of separation techniques have been coupled with MALDI-MS analysis. Liang, et al., reported the use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins prior to application of the samples for evaporation and crystallization for MALDI-MS analysis. Anal. Chem. 68: 1012-1018 (1996). Rahbek-Nielsen, et al., used high performance liquid chromatography to separate peptides prior to application of peptide fractions onto a probe tip which had been precoated with matrix. J. Mass Spec. 32: 943-958 (1997). Zhang and Caprioli produced off-line coupling of capillary electrophoresis (CE) with MALDI-MS by fixing a cellulose membrane onto a polished MALDI sample plate, with the plate mounted on the moveable stage with the exit end of the capillary contacting the membrane strip. The target plate was moved 1.5 to 2.0 minutes after the start of the CE analysis and movement continued until 3.3 minutes later. During this time the stage was moved approximately 5 cm, with continuous deposition of sample along the sample track. The target plate was then removed and placed in the mass spectrometer. J. Mass. Spec. 31(9): 1039-1046 (1996).
Hahner, et al., describe using MALDI-MS for direct sequencing of RNA by enzymatically cleaving the RNA molecules prior to placing 0.6 ml aliquots of analyte solution on a flat inert metal substrate for analysis. Nuc. Acids Res. 25: 1957-1964 (1977). Beavis, et al., U.S. Pat. No. 5,288,644, describe the use of MALDI-MS with standard DNA sequencing techniques to perform DNA sequencing. The four separate collections of DNA fragments are applied to a MALDI-MS probe, along with matrix solution. The volatile solvents are then removed by room temperature evaporation before MALDI-MS analysis. Jurinke, et al., describe the use of nested PCR and MALDI-MS analysis to sequence viral DNA. Nested PCR samples are prepared from the viral DNA. Half a microliter of the PCR sample is then pipetted onto a MALDI-MS sample holder, where it is mixed with an equal volume of matrix solution and dried at ambient temperature before being placed into the mass spectrometer. Gen. Anal. Biomolecular Eng. 13: 67-71 (1996).
Each of the previously mentioned techniques, however, is limited to off-line coupling of the separation system with MALDI-MS. Preparation of sample for MALDI-MS analysis has posed problems for on-line coupling of sample preparation and analysis.
MALDI analysis is accomplished by mixing analyte with a matrix solution consisting of a suitable small organic acid, such as 2,5-dihydroxybenzoic acid or a-cyano-4-hydroxycinnamic acid, where the matrix is presumed to isolate the biopolymer molecules from each other, absorb energy from the laser light, and promote efficient analyte ionization in a gas phase. Sample preparation has been most commonly done by mixing suitable proportions of analyte and matrix solutions, then drying an aliquot of the mixture onto a probe made of a solid material such as an inert metal. MALDI sample preparation has therefore proven unacceptable for on-line use with liquid capillary separation systems.
Once the sample has been crystallized on the probe, a laser beam directed to the sample provides energy to desorb matrix and analyte and to obtain efficient ionization in a gas phase as proton transfer from the small organic acid matrix molecules to analyte molecules occurs without decomposing the analyte molecules. The host matrix is selected to absorb the radiation, and therefore the wavelength of the radiation is selected according to the absorbance characteristics of the matrix material. In a TOF mass spectrometer, the mass of the ionized analyte molecule can than be determined by the arrival time of an individual analyte ion at the detector, a function of the mass/charge ratio.
On-line coupling of separation system with MALDI-MS has been attempted using laser energy directed to an aerosol suspension of analyte/matrix molecules. This technique, however, has proven unsuccessful at providing the same quality results as solid-state sample preparation methods in off-line coupled systems.
U.S. Pat. No. 5,643,800 describes a method of off-line sample preparation to produce a more homogeneous sample, which has been shown to improve sensitivity and resolution of MALDI-TOF analysis. Anal. Chem. 66: 3281-3287 (1994). A mixture of analyte and matrix is sprayed onto a probe tip, where it is crystallized by lyophilization to form a homogeneous analyte/solvent mixture. The probe tip is then removed and placed into a mass spectrometer for analysis.
U.S. Pat. No. 5,499,902 describes an alternative means for directly connecting an analytical column with a mass spectrometer, comprising four or more trapping columns for washing, trapping, and desalting the component of interest, as well as for introducing the sample into the mass spectrometer. Although the method promotes automation of sample preparation and analysis in a mass spectrometer, the additional step of mixing analyte and matrix, followed by evaporation and crystallization of the sample on a sample probe or sample surface, would be too time-consuming for sample preparation and analysis of samples from a liquid capillary separation system.
What is needed then, and what the present invention provides, is a system and method for preparation of a homogenous sample to be deposited on a solid surface for MALDI-MS analysis which can produce sample deposit on and sample desorption/ionization from the solid surface at a rate that would allow for analysis of continuously eluting sample from a liquid capillary separation system.